Please share the protocol for antigen uptake assay by cancer cells. I want to use Dextran-FITC to track the endocytosis by cancer cells. If anyone has the protocol or any suggestions kindly share.
Not of Dextran-FITC but I have experience of FITC ! and not for Endocytosis.
I have experience using it on macrophages.
I can suggest this briefly.
First, treat the cells as you have planned. Then, wash the cells with a suitable buffer (PBS for example). Dilute the FITC-Dextran beads in serum-free media and add it to the cells. Remember to have a negative control where you will put the cells at 4 degrees Celsius for the beads to adhere, but not be engulfed. Incubate the cells of the sets of interest at 37 degrees Celcius with beads in serum-free media for 45 minutes. Wash all the sets thoroughly with PBS and record the data (flow cytometry may be a good choice, although you may opt for fluorescence plate reading or more preferably confocal fluorescence imaging).
Note: The dilution of FITC-Dextran beads is subjected to be optimized. Take a range of dilutions (such as 1:100 to 1:1200) and choose the best shift in peak (doable in flow cytometry). Be careful to wash the beads out thoroughly to get better results.
Hope it helps you. Wish for great results in your experiment. Please recommend it if it works. Share with your teammates. Thank you.
Another protocal I used before.
1.Quantitative dextran by measure fluorescence intensity of FITC.
2.Wash your cells in 24/96 well plates and incubate them with your samples with different concentration like 5 or 10 ug/mL.
3.After 1h, remove the medium with samples and wash twice by PBS.
4.Lysis your cell by 0.1% TritonX-100 30min, measure the fluorescence intensity of FITC.
Hope it helpful.
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